The study and genetic manipulation of plants has a long history that began even before the famed studies of Gregor Mendel in the 19th century. In perfecting this science, scientists have accomplished modification of particular traits in plants ranging from potato tubers having increased starch content to oilseed plants such as canola and sunflower having increased or altered fatty acid content. With the increased consumption and use of plant oils, the modification of seed oil content and seed oil levels has become increasingly widespread. The seed oil production or composition has been altered in numerous traditional oilseed plants such as soybean (U.S. Pat. No. 5,955,650), canola (U.S. Pat. No. 5,955,650), sunflower (U.S. Pat. No. 6,084,164) and rapeseed (Toepfer et al. 1995, Science 268:681-686), as well as non-traditional oil seed plants such as tobacco (Cahoon et al. 1992, Proc. Natl. Acad. Sci. USA 89:11184-11188).
Plant seed oils comprise both neutral and polar lipids (see Table 1). The neutral seed lipids contain primarily triacylglycerol, which is the main storage lipid that accumulates in oil bodies in seeds. The polar lipids are mainly found in the various membranes of the seed cells, e.g. the endoplasmic reticulum, microsomal membranes and the cell membrane. The neutral and polar lipids contain several common fatty acids (see Table 2) and a range of less common fatty acids. The fatty acid composition of membrane lipids is highly regulated and only a select number of fatty acids are found in membrane lipids. On the other hand a large number of unusual fatty acids can be incorporated into the neutral storage lipids in seeds of many plant species (Van de Loo, F. J. et al. 1993, Unusual Fatty Acids in Lipid Metabolism in Plants pp 91-126 editor T S Moore Jr. CRC Press).
Lipids are synthesized from fatty acids and their synthesis may be divided into two parts: the prokaryotic and the eukaryotic pathway (J Ohlrogge & J Browse 1995, Lipid Biosynthesis Plant Cell 7:957-970). The prokaryotic pathway is located in plastids that are the primary site of fatty acid biosynthesis. Fatty acid synthesis begins with the conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (ACCase). Malonyl-CoA is converted to malonyl-ACP by the malonyl-CoA:ACP transacylase. The enzyme beta-keto-acyl-ACP-synthase III (KAS III) catalyzes a condensation reaction in which the acyl group from acetyl-CoA is transferred to malonyl-ACP to form 3-ketobutyryl-ACP. In a subsequent series of condensation, reduction and dehydration reactions the nascent fatty acid chain on the ACP cofactor is elongated by the step-by-step addition (condensation) of two carbon atoms donated by malonyl-ACP until a 16- or 18-carbon saturated fatty acid chain is formed. The plastidial delta-9 acyl-ACP desaturase introduces the first unsaturated double bond into the fatty acid. Thioesterases cleave the fatty acids from the ACP cofactor and free fatty acids are exported to the cytoplasm where they participate as fatty acyl-CoA esters in the eukaryotic pathway. In this pathway the fatty acids are esterified by glycerol-3-phosphate acyltransferase and lysophosphatidic acid acyltransferase to the sn-1 and sn-2 positions of glycerol-3-phosphate, respectively, to yield phosphatidic acid (PA). The PA is the precursor for other polar and neutral lipids, the latter being formed in the Kennedy pathway (Shanklin and Cahoon 1998, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:611-641; Voelker 1996, Genetic Engineering ed.: J K Setlow 18:111-13; Frentzen 1998, Lipid 100(4-5):161-166).
Acetyl-CoA in the plastids is the central precursor for lipid biosynthesis. Acetyl-CoA can be formed in the plastids by different reactions and the exact contribution of each reaction is still being debated (J Ohlrogge & J Browse 1995, Lipid Biosynthesis Plant Cell 7:957-970). It is however accepted that a large part of the acetyl-CoA is derived from glucose-6-phospate and pyruvate (i.d. phosphoenolpyruvate) that are imported from the cytoplasm into the plastids. Sucrose is produced in the source organs (leaves, where photosynthesis takes place) and is transported to the developing seeds that are also termed sink organs. In the developing seeds, the sucrose is the precursor for all the storage compounds, i.e. starch, lipids and partly the seed storage proteins. Therefore, it is clear that carbohydrate metabolism in which sucrose plays a central role is very important to the accumulation of seed storage compounds.
Although lipid and fatty acid content of seed oil can be modified by the traditional methods of plant breeding, the advent of recombinant DNA technology has allowed for easier manipulation of the seed oil content of a plant, and in some cases, has allowed for the alteration of seed oils in ways that could not be accomplished by breeding alone. For example, introduction of a Δ12-hydroxylase nucleic acid sequence into transgenic tobacco resulted in the introduction of a novel fatty acid, ricinoleic acid, into the tobacco seed oil (Van de Loo, et al. 1995, Proc. Natl. Acad. Sci USA 92:6743-6747). Tobacco plants have also been engineered to produce low levels of petroselinic acid by the introduction and expression of an acyl-ACP desaturase from coriander (Cahoon et al. 1992, Proc. Natl. Acad. Sci USA 89:11184-11188).
The modification of seed oil content in plants has significant medical, nutritional and economic ramifications. With regard to the medical ramifications, the long chain fatty acids (C18 and larger) found in many seed oils have been linked to reductions in hypercholesterolemia and other clinical disorders related to coronary heart disease (Brenner R. R. 1976, Adv. Exp. Med. Biol. 83:85-101). Therefore, consumption of a plant having increased levels of these types of fatty acids could reduce the risk of heart disease. Enhanced levels of seed oil content also increase large-scale production and thereby reduce the cost of these oils.
In order to increase or alter the levels of compounds such as seed oil in plants, nucleic acid sequences and proteins regulating lipid and fatty acid metabolism must be identified. As mentioned earlier, several desaturase nucleic acids such as the Δ6-desaturase nucleic acid, Δ12-desaturase nucleic acid and acyl-ACP desaturase nucleic acid have been cloned and demonstrated to encode enzymes required for fatty acid synthesis in various plant species. Oleosin nucleic acid sequences from such different species as Brassica, soybean, carrot, pine and Arabidopsis have also been cloned and determined to encode proteins associated with the phospholipid monolayer membrane of oil bodies in those plants.
Although several nucleic acids that are involved in enzymatic steps of the metabolism of lipids, fatty acids and starches have been cloned and identified, there are likely a multitude of such plant nucleic acids that have yet to be identified. Phenotypic analysis of several oilseed plants and other mutated plants has revealed other putative proteins involved in plant lipid metabolism, but the prior art has yet to describe the genomic location of these proteins or the nucleic acids that encode them.
An exemplary study is that of the oilseed plant Arabidopsis thaliana. In 1998, Focks and Benning isolated and characterized a wrinkled mutant of Arabidopsis thaliana designated wri1 (Plant Physiology 1998, 118:91-101). The wri1 mutant has a decreased seed oil content that was speculated to be due to a defect in the seed-specific regulation of carbohydrate metabolism. In the wri1 mutant, the activities of several glycolytic enzymes were reduced and the mutant seeds were impaired in the incorporation of sucrose and glucose into triacylglycerol lipids, while important precursor molecules for plastidial lipid biosynthesis, like pyruvate and acetate, were incorporated at increased rates. This biochemical evidence was interpreted by Focks and Benning as indication that the WRI1 protein could be a regulatory protein governing carbohydrate metabolism during seed development or a hexokinase that may act as a sugar sensor in developing seeds, and thus controlling the activities of several glycolytic enzymes (Plant Physiology 1998, 118:91-101). The wri1 phenotype (wrinkled seeds) has been found in two different allelic Arabidopsis thaliana mutants, namely wri1-1 and wri1-2.
Since the discovery of the wri1 phenotype by Focks and Benning, the Arabidopsis thaliana genome was sequenced in its entirety (The Arabidopsis Genome Initiative 2000 Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796-844). These genomic sequences have been annotated and open reading frames encoding putative proteins have been assigned in an automatic process. Importantly however, this annotation is only based upon homologies with other sequences with known functions, and therefore, in no way identifies the true location, sequence or functionality of an Arabidopsis thaliana nucleic acid sequence. The annotation and assignment of open reading frames also in no way describes the location, function or sequence of the wri1 gene.
Therefore, what is needed in the art is the elucidation of the location and identity of the one or more nucleic acids associated with the wri1 mutation in Arabidopsis thaliana along with an understanding of the functionality of the proteins and protein fragments encoded by those nucleic acids.